Clinical, Virologic, and Immunologic Evaluation of Symptomatic Coronavirus Disease 2019 Rebound Following Nirmatrelvir/Ritonavir Treatment

Abstract

Background: Nirmatrelvir/ritonavir, the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease inhibitor, reduces the risk of hospitalization and death by coronavirus disease 2019 (COVID-19) but has been associated with symptomatic rebound after therapy completion.

Methods: Six individuals with relapse of COVID-19 symptoms after treatment with nirmatrelvir/ritonavir, 2 individuals with rebound symptoms without prior antiviral therapy and 7 patients with acute Omicron infection (controls) were studied. Soluble biomarkers and serum SARS-CoV-2 nucleocapsid protein were measured. Nasal swabs positive for SARS-CoV-2 underwent viral isolation and targeted viral sequencing. SARS-CoV-2 anti-spike, anti-receptor-binding domain, and anti-nucleocapsid antibodies were measured. Surrogate viral neutralization tests against wild-type and Omicron spike protein, as well as T-cell stimulation assays, were performed.

Results: High levels of SARS-CoV-2 anti-spike immunoglobulin G (IgG) antibodies were found in all participants. Anti-nucleocapsid IgG and Omicron-specific neutralizing antibodies increased in patients with rebound. Robust SARS-CoV-2-specific T-cell responses were observed, higher in rebound compared with early acute COVID-19 patients. Inflammatory markers mostly decreased during rebound. Two patients sampled longitudinally demonstrated an increase in activated cytokine-producing CD4+ T cells against viral proteins. No characteristic resistance mutations were identified. SARS-CoV-2 was isolated by culture from 1 of 8 rebound patients; Polybrene addition increased this to 5 of 8.

Conclusions: Nirmatrelvir/ritonavir treatment does not impede adaptive immune responses to SARS-CoV-2. Clinical rebound corresponds to development of a robust antibody and T-cell immune response, arguing against a high risk of disease progression. The presence of infectious virus supports the need for isolation and assessment of longer treatment courses.

Clinical trials registration: NCT04401436.

Keywords: COVID-19; COVID-19 rebound; COVID-19 transmission; antiviral therapy; nirmatrelvir/ritonavir.

Figure 1.

Comparison of clinical laboratory and virologic measurements across the groups. Lines represent median and points represent individual results. The 2 longitudinal patients are identified by an open triangle and open diamond, respectively. The open square represents the coronavirus disease 2019 (COVID-19) rebound patients who did not receive nirmatrelvir-ritonavir. Clinical values for C-reactive protein (A), absolute neutrophil count (B), and absolute lymphocyte count (C) across the acute, rebound, and late presenting COVID-19 cohorts. Severe acute respiratory syndrome coronavirus 2 cycle threshold from nasal swab samples (D) and serum nucleocapsid Ag (E). Cycle threshold was not available at rebound time point for longitudinal patient 1 (diamond) as it was run on a BioFire platform. Abbreviation: Ag, antigen.

Figure 2.

Viral sequencing of severe acute respiratory syndrome coronavirus 2 isolated from acute and rebound groups compared with Omicron BA.2 subvariant. A, Nucleotide mutations in the sequenced isolates from the acute and rebound groups compared with Omicron BA.2 subvariant. Vertical dashes for each isolate correspond to changes from the Omicron BA.2 subvariant. Zoomed images of the nsp5 region and the spike region are shown from isolates from the acute group (HES-CS-9, HES-CS-154, HES-CS-124, and HES-CS-151-1) and rebound group (HES-CS-144-2, HES-CS-147, HES-CS-140, HES-CS-150, and HES-CS-151-2) with specific mutations labeled. B, Longitudinal sequencing data for patient 1. Abbreviations: aa, amino acid; nt, nucleotide.

Figure 3.

Comparison of antibody level measurements across the groups. Antibody levels by enzyme-linked immunosorbent assay (ELISA) against the spike protein (A), spike–receptor binding domain (RBD) (B), and the nucleocapsid protein (C) presented as OD. ELISA data not available for longitudinal patient 1 (diamond), 1 acute patient, and 1 rebound without nirmatrelvir-ritonavir patient (square). sVNT to detect neutralizing antibodies against the wild-type (D) and Omicron (E) spike protein presented as percent binding inhibition. Dotted lines represent the cutoff for a positive result for the antibody tests (A–C). Mann–Whitney test was used to derive P values comparing the acute and rebound coronavirus disease 2019 cohorts. Abbreviations: Ig, immunoglobulin; OD, optical density; RBD, receptor-binding domain; sVNT, surrogate viral neutralization test.

Figure 4.

Comparison of CD4⁺ T-cell responses across the groups and longitudinal immune responses of the 2 patients with sampling at acute and rebound time points. Absolute T-cell counts compared across groups (A, B). Lines represent median values and points represent individual results. The 2 longitudinal patients are identified by an open triangle and open diamond. The empty square represents the coronavirus disease 2019 rebound patient who did not receive nirmatrelvir-ritonavir. T-cell subset flow cytometry data not available for longitudinal patient 2 (triangle) at rebound time point. T-cell stimulations were performed with peptide pools corresponding to spike, nucleocapsid, and membrane proteins as listed on the x-axis. Bars represent medians and groups are defined as acute, rebound, and late presentations. Severe acute respiratory syndrome coronavirus 2–specific CD4 T-cell responses are highlighted by memory (C), cytokine-producing (CD154 + IFN-γ+, CD154 + TNF-α+ or CD154 + IL-2+) (D), activated (CD154 + CD69+) (E), or Ag-specific proliferating (Ki-67+) and activated (PD-1+) T cells (F). For phenotyping of Ki-67 + and PD-1+ cells, a threshold of at least 20 events and a 2-fold increase over unstimulated cells was used, and samples were excluded if they did not meet these thresholds (E, F). Serum N Ag and C-reactive protein trends from the 2 longitudinal patients (G, H). T-cell responses and neutralizing antibodies from the acute and rebound presentation for 2 patients with longitudinal samples (I, J). T-cell responses are from S and N stimulations. Ag-specific CD4 T cells defined by (CD154 + CD69+, CD154 + IFN-γ+ and CD154 + TNF-α+), and neutralizing antibodies represented by percent binding inhibition on the sVNT. Abbreviations: Ag, antigen; S, spike; N, nucleocapsid; spec, specific; sVNT, surrogate virus neutralization test.

Figure 5.

Innate and adaptive biomarkers across study groups compared with healthy controls and heat map of data from study participants. Lines represent medians and points represent individual results across the acute, rebound, and late presentation coronavirus disease 2019 (COVID-19) clinical groups compared with healthy control population (HC). Healthy controls consisted of 5 women and 2 men with a median age of 59 years (range, 45–66). These samples were unmatched and are included to provide a baseline range for these biomarkers in an otherwise healthy population. The 2 longitudinal COVID-19 rebound patients are identified by an open diamond (patient 1) and open triangle (patient 2). The open square represents the COVID-19 rebound patient who did not receive nirmatrelvir-ritonavir (NMV-r). A, Innate biomarkers (IL-6, IL-8, TNF-α, CXCL-10, sCD14, and IL-18BP) classically increased in acute COVID-19 are downtrending at time of rebound. B, Adaptive biomarkers representing T-cell activation (IFN-γ, CXCL-9, sCD25) were stable or increasing at rebound consistent with a developing T-cell response. C, Comprehensive heat map with unsupervised clustering of variables including clinical laboratory tests, virologic measurements, biomarkers, and profiling of adaptive responses identified that all patients with rebound COVID-19 after NMV-r form a unique cluster distinct from those with acute infection. The late presenting patient and rebound patient without NMV-r cluster with the other rebound COVID-19 patients. Analysis performed in R using the pheatmap package. One patient with acute COVID-19 and 1 rebound patient without NMV-r (both with BA.5) were excluded from the heat map due to missing biomarker data. Abbreviations: CXCL-10, chemokine (C-X-C motif) ligand 10; HC, healthy controls; IFN-γ, interferon γ; IL, interleukin; IL-18BP, interleukin 18 binding protein; sCD25, soluble CD25; TNF-α, tumor necrosis factor α.